There is an expanding need for specific, sensitive, rapid and cost effective methods of detecting and quantifying chemical, biological and biochemical substances such as antibodies, hormones, viruses, enzymes, metabolites, narcotics, poisons, drugs, microorganisms and nucleic acids. The required sensitivity and specificity are obtained by using binding reactions, e.g., antigen-antibody reactions where the presence of the complex of diagnostic value i.e., analyte, is indicated by means of a detectable label attached to one or more of the complexing materials. An example of commercially useful labeling compounds are those capable of generating a luminescence based on the photochemical, chemical or electrochemical excitation methods.
The analytical methods based on luminescence in its various modifications are generally known for their sensitivity, but each have their own shortcomings at very low concentrations of the emitting species. The sensitivity of fluorescence is limited by Raleigh and Raman scattering phenomena and as well as fluorescent impurities which increase the non-specific background emission. Phosphorescence is mainly restricted to solid state and the emission from those very few compounds which have room temperature phosphorescence in solution is generally extremely sensitive to oxygen, which hampers their practical applications. The method based on conventional fluorescence and phosphorescence use an excitation by light and need an appropriate light source and optics. The methods based on chemiluminescence do not need excitation optics and the instrumentation is generally very simple. However, chemiluminescence methods are often subjects to serious chemical interference. The method based on an instrumentally simple electrochemical excitation (i.e., electrogenerated chemiluminescence or ECL) utilizes an excitation by an electrical pulse applied to an electrode which provides a low detection limit.
ECL of inorganic and organic compounds in electrolyte solutions is well known in the art. For instance, the anodic ECL of luminol at the platinum electrode in an aqueous electrolyte has been studied since 1929 (for instance, N, Harvey, J. Phys. Chem. 33 (1929) 1456; K. Haapakka and J. Kankare, Anal. Chim. Acta 138 (1982) 263), an anodic ECL of Ru(bpy).sub.3.sup.2+ in the presence of oxalate in an aqueous electrolyte and a cathodic ECL of Ru(bpy).sub.3.sup.2+ in the presence of peroxydisulfate in an acetonitrile/water mixture have been reported by Bard et al. (D. Ege, W. Becker and A. Bard, Anal. Chem. 56 (1984) 2413), a cathodic ECL of numerous inorganic ions (K. Haapakka, J. Kankare and S. Kulmala, Anal. Chim. Acta 171 (1985) 259) and numerous organic compounds (K. Haapakka, J. Kankare and 0. Puhakka, Anal. Chim. Acta 207 (1988) 195) at an oxide-covered aluminium electrode in aqueous electrolytes containing an appropriate oxidizing agent have been reported, where the short-lived ECL is measured during the electric pulse applied to the electrodes. A variety of terbium(III) complexes are capable of initiating a long-lived terbium(III)-specific cathodic ECL at an oxide-covered aluminium electrode in an aqueous electrolyte containing peroxydisulfate, which makes possible to eliminate the short-lived background ECL by using a time-resolved ECL detection thus creating the basis for extreme trace analysis (J. Kankare, K, Falden, S. Kulmala and K. Haapakka, Anal. Chim. Acta 256 (1992) 17; J. Kankare, A. Karppi and H. Takalo, Anal. Chim. Acta 295 (1994) 27). Binding assays of the analytes of interest based on the measurement of ECL at the electrode surface have been proposed: for instance, A. Bard et al. (D. Ege, W. Becker and A. Bard, Anal. Chem. 56 (1984) 2413 and WO 86/02734) have suggested ruthenium(III)- and osmium(III)-containing ECL labels; J. Kankare and K. Haapakka (GB 2217007 B, U.S. Pat. No. 5,308,754), J. Kankare, K. Haapakka, S. Kulmala, V. Nanto, J. Eskola and H. Takalo, Anal. Chim. Acta 266 (1992) 205 and M. Billadeau et al. (WO 96/41177) have suggested the use of Ln(III)-containing ECL labels (Ln(III).dbd.Dy(III), Eu(III), Sm(III), Tb(III) in the binding assays based on the time-resolved ECL. Numerous sample cell configurations and methods of measurement for the ECL detection have been proposed where the ECL is generated either at the surface of the electrode (for instance in EP 65 8760 A1 and WO 96/28538) or at the surface of magnetic beads collected onto the surface of the electrode (for instance in NVO 92/14139; WO 92/14138, JP 08190801 A2 and WO 96/15440). The ECL detectors have been applied in High Pressure Liquid Chromatography (for instance, D. Skotty, W. Lee and T. Nieman, Anal. Chem. 68 (1996 1530) and in Capillary Electrophoresis (for instance, G. Forbes, T. Nieman and J Sweedler, Anal. Chim. Acta 347 (1997) 289).
Typically for ECL, the luminescent compound must be in the close proximity of the electrode surface. Especially as to the ECL immunoassays, the ECL label, which contains luminescent compound attached to antibody or antigen, is bound to the electrode surface, e.g., by the direct immunoreaction where one of the immunoreagents is immobilized on the electrode surface (J. Kankare and K. Haapakka, GB 2217007 B, U.S. Pat. No. 5,308,754), or indirectly by utilizing non-conducting magnetic beads coated with immunoreagents, which after the immunoreaction has occurred, are collected at the electrode surface by a magnetic field. The advantage of using these magnetic beads is a considerably more efficient binding reaction as compared to that, e.g., in the antibody-coated planar electrode surface. However, use of magnetic beads in ECL assays are not without disadvantages: (i) the excitation efficiency is often low because of the an excitation distance of around 25 .ANG. or less from the electrode surface is required (see, e.g., WO 92/14139): (ii) the efficiency of detecting the emitted light from the proximity of the electrode surface is hindered by the light-shielding bead layer and (iii) the magnetic beads detach from the electrode surface during the short excitation pulse thereby inhibiting excitation efficiency.
While magnetic beads do provide an improvement over conventional ECL methods, there is still a need in the art for ECL assays that exhibit further increased sensitivity. Accordingly, it is an object of the present invention to provide an ECL method having increased sensitivity.